In most processes within molecular biology it is critical to have a reaction that takes into account the ability to detect the occurrence of a particular event. For instance, events such as the incorporation of one, or many, nucleotides onto an extension primer may be indicative of the presence of a single nucleotide polymorphism. Upon the occurrence of an event, such as the incorporation of one or more nucleotides, a mechanism for detection may be built into the reaction or, alternatively, used in a subsequent reaction to provide a means to signal the occurrence of the event.
Single base chain extension, whereby the incorporation of a single di-deoxy nucleotide, which may contain a dye for detection (or use mass as a means for detection), is an example of a reaction that contains both an interrogation and a detection mechanism for nucleic acids. One of the simplest ways to detect a single nucleotide extension is by fluorescence, for example, by using fluorescently labeled nucleotides or a FRET (fluorescence resonance energy transfer) signal. However, single base chain extension is costly because it requires the use of fluorescently labeled nucleotides, and/or probes. Additionally, the concentration of both the probes and the nucleotides must be high for the reaction to work, resulting in a high background signal. Consequently, multiple rigorous wash steps must be employed to remove the unhybridized or unbound material, which is not practical for most applications, particularly when using small reaction vessels.
Another rendition of fluorescence-based detection is the utilization of a fluorescently tagged molecule (a fluorophore) and a quencher. As described above, when working with a plurality of molecules, the concentration of the fluorescence molecules may be problematic in terms of signal.
One solution to this problem is the use of double stranded fluorescence-quencher probes. Such assays are often optimized for specific parameters such as probe length, target DNA length, or enzymatic reaction. Most fluorophore-quencher pair assays are effective only for short DNA strands or amplicons (<200 bp) under tight thermal control (e.g., Holland et al., “Detection of specific polymerase chain reaction product by utilizing the 5′→3′ exonuclease activity of Thermus aquaticus DNA polymerase”, PNAS (1991) vol. 88, pp. 7276-7280; Piatek, et al., “Molecular beacon sequence analysis for detecting drug resistance in Mycobacterium tuberculosis”, Nat Biotechnol (1998) vol. 16, no. 4, pp. 359-363; Udvardi, et al., “Eleven golden rules of quantitative RT-PCR”, The Plant Cell (2008) vol. 20, pp. 1736-1737; Vet al., “Design and Optimization of Molecular Beacon Real-Time Polymerase Chain Reaction Assays”, In: P. Herdewijn, ed. 2004. Oligonucleotide Synthesis: Methods and Applications (Methods in Molecular Biology, vol. 288). New Jersey: Humana Press Inc., pp. 273-290; “Top Ten Pitfalls in Quantitative Real-time PCR Primer/Probe Design and Use”, Applied Bio systems TechNotes (2011) vol. 13, no. 4 (www.ambion.com/techlib/tn/134/13.html); “PCR Primer Design Guidelines”, PREMIERBiosoft (2011) (www.premierbiosoft.com/tech_notes/PCR_Primer_Design.html).
Accordingly, there is a need for a reliable, efficient and cost-effective method for detecting the presence or absence of a particular nucleic acid sequence using a variety of nucleic acid probe lengths, target nucleic acid lengths, temperature conditions or DNA polymerases, as provided by the following invention.
Citation or identification of any document in this application is not an admission that such document is available as prior art to the present invention.